Visualization of micromorphology of leaf epicuticular waxes of the rubber tree Ficus elastica by electron microscopy

Abstract

Ultrastructural aspects of leaf epicuticular waxes were investigated in Ficus elastica by scanning and transmission electron microscopy. Glossy leaves of the rubber tree were collected and subjected to different regimes of specimen preparation for surface observations. F. elastica leaves were hypostomatic and stomata were surrounded with a cuticular thickening that formed a rim. The most prominent epicuticular wax structures of F. elastica leaves included granules and platelets. Without fixation and metal coating, epicuticular wax structures could be discerned on the leaf surface by low-vacuum (ca. 7 Pa) scanning electron microscopy. In terms of delineation and retention of the structures, the combination of vapor fixation by glutaraldehyde and osmium tetroxide with subsequent gold coating provided the most satisfactory results, as evidenced by high resolution and sharp protrusions of epicuticular waxes. However, erosion of epicuticular wax edges was noted in the immersion fixed leaves, showing less elongated platelets, less distinct wax edges, and granule cracking. These results suggest that the vapor fixation procedure for demonstrating three-dimensional epicuticular wax structures would facilitate characterization of diverse types of waxes. Instances were noted where epicuticular waxes grew over neighboring epidermal ridges and occluded stomata. In cross sections, epicuticular waxes were observed above the cuticle proper and ranged approximately from 100 nm to 500 nm in thickness. The native leaf epicuticular waxes had many layers of different electron density that were oriented parallel to each other and parallel or perpendicular to the cuticle surface, implying strata of crystal growth. Such retention of native epicuticular wax structures could be achieved through the use of acrylic resin treated with less harsh dehydrants and mild heat polymerization, alleviating wax extraction during specimen preparations.